Fungal degradation and bioremediation system for pentachlorophenol-treated wood

ABSTRACT

A method for degrading and/or bioremediating waste wood containing pentachlorephenol (penta) using a fungal inoculum is disclosed. The fungal inoculum comprises of at least one penta-tolerant fungi, a lignocellulosic substrate and a nutrient supplement. The fungal inoculum is applied to the waste wood and maintained in an aerated and hydrated environment having temperature conditions sufficient to allow the inoculum to grow and metabolize the pentachlorophenol. The inoculum and the waste wood are combined until an end product is achieved that is at least partially remediated or of a reduced volume.

CROSS-REFERENCE TO RELATED APPLICATIONS STATEMENT REGARDING FEDERALLYSPONSORED RESEARCH OR DEVELOPMENT BACKGROUND OF THE INVENTION

[0001] The present invention relates to fungal inocula and their use indegrading and bioremediating wood treated with chemical preservatives.More specifically, this invention discloses and claims a fungalinoculum, the method of its preparation, and its use in degrading andbioremediating wood treated with pentachlorophenol.

[0002] Wood used in the construction of today's decks, docks andbuildings, or as utility poles and railroad ties, is typically treatedwith a chemical preservative to prevent its deterioration and extend itsservice life. The chemical preservative used will generally depend uponthe intended use of the wood and often includes chemicals such aspentachlorophenol (penta), chromated copper arsenate (CCA), ammoniacalcopper quat (ACQ), and creosote. Until its use was restricted in 1986,penta was typically used to treat aboveground lumber, timber, bridgeties and mine ties located in non-salt water environments.

[0003] The disposal of penta-treated wood once it reaches the end of itsuseful life requires careful consideration because of its toxicity. Forexample, penta-treated wood is generally not burned in open fires oropen stoves, fireplaces or residential boilers because of the toxicchemicals which may be produced as part of its smoke and ash.

[0004] Accordingly, penta-treated wood is typically collected and storedat landfills or other facilities where large quantities of waste wood isaccumulating at an alarming rate. The collection and storage of suchlarge quantities of waste wood creates an environment where thecontamination of surrounding soil and groundwater by toxic,environmentally-persistent chemicals is a likely result.

[0005] Contamination of soils and groundwater with toxic,environmentally-persistent chemicals is a serious problem. Toxic,environmentally-persistent chemicals are those that are resistant todegradation in the natural environment. As such, these chemicals pose amulti-faceted problem in that as they persist and accumulate in theenvironment, their toxicity, including in many instances, provencarcinogenicity, presents substantial health risks to both animals andhuman beings. Environmental contamination from penta-treated wood is aspecific concern in view of the large volume of penta-treated waste woodexpected to be removed from service and disposed of in the near future.

[0006] The prior art is replete with methods for degrading hazardouschemicals. However, this prior art is generally, and specifically,directed towards halogenated aromatic compounds. Suggested treatmentstrategies include incineration in commercial or industrial incineratorsunder federal and state regulation, removal and isolation of thecontaminated materials, and degradation of the pollutant by bacteria.

[0007] All of these strategies suffer from serious deficiencies.Incineration is extremely expensive due to the required energy andsafety expense and the necessity of moving the contaminated material toremote locations. Incineration is also impractical because of the largequantities of waste which needs processing. Removal and isolation of thecontaminated material is also expensive and does nothing to effect along-term solution. Degradation of the chemicals using bacteria has alsoproven ineffective due to the bacteria's specificity for particularchemicals and its sensitivity to the toxic chemicals and environmentalconditions.

[0008] U.S. Pat. No. 5,476,788 employs another strategy which utilizesan inoculum containing the lignin-degrading fungal species Phanerochaetechrysosporium, Phanerochaete sordida, or Trametes hirsuta to remediatesolid materials, such as soils, sludge, sediments, and debris (e.g.,woods), contaminated with pentachlorophenol. The inoculum contains oneor more of the fungal strains and a lignocellulosic substrate, i.e.,sawdust. In its use, the inoculum is combined with thepentachlorophenol-contaminated material and the entire mixture isaerated and hydrated until the inoculum metabolizes thepentachlorophenol to a less toxic product. Typically, this less toxicproduct includes pentachloroanisole.

BRIEF SUMMARY OF THE INVENTION

[0009] The present invention is summarized in that it discloses a fungalinoculum, the method of its preparation, and its use in degrading and/orbioremediating wood treated with the chemical preservativepentachlorophenol (penta).

[0010] The disclosed fungal inoculum generally comprises of at least onepenta-tolerant fungus, a lignocellulosic substrate, and a nutrientsupplement. The penta-tolerant fungal strains are preferably selectedfrom the group consisting of Meruliporia incrassata (Mad-563), Antrodiaradiculosa (FP-103272-sp), Antrodia radiculosa (L-11659-sp) and Antrodiaradiculosa (FP-90848-T). The lignocellulosic substrate is preferablysawdust or wood chips. Other feasible substrates are rice straw, cornstalks, and wheat straw. The nutrient supplement is preferably selectedfrom the group consisting of corn steep liquor, cornmeal and wheatbran.

[0011] In one preferred embodiment, the fungal inoculum is prepared byfirst growing the penta-tolerant fungus in dark, aerobic conditions,having a relative humidity and a temperature sufficient to supportfungal growth. The fungus is then combined with a homogenous matrixcomprising sterile water, the nutrient supplement, and the sterilizedlignocellulosic substrate, to form the fungal inoculum. The fungalinoculum is then allowed to mature by exposing the mixture to dark,aerobic conditions, at a relative humidity and in a temperature rangesufficient to allow the fungus to reach a confluent growth.

[0012] Waste wood containing penta is remediated or degraded byinoculating the waste wood in the fungal inoculum. To inoculate thewaste wood, the fungal inoculum is first spread over the waste wooduntil all of the waste wood is covered. The waste wood and fungalinoculum mixture is then aerated and hydrated for a time and underconditions sufficient to allow the inoculum to at least partiallyremediate the penta and/or degrade the waste wood to a desireddegradation product. In some instances, the degradation product may becapable of reuse in paper or wood composites, or simply have a reducedvolume.

[0013] It is an object of the present invention to provide a method forbioremediating and/or degrading chemically treated waste wood to achievea product having a reduced volume and/or the capacity to be reused as awood fiber resource.

[0014] It is another object of the present invention to provide a fungalinoculum, and a method for preparing said fungal inoculum, which isuseful in bioremediating and/or degrading chemically treated waste wood.

[0015] It is another object of the present invention to provide a fungalinoculum, and a method for using the fungal inoculum, to degrade and atleast partially remediate waste wood chemically treated withpentachlorophenol.

[0016] One advantage of the present invention is that the penta-tolerantfungi utilized do not require genetic alteration to specifically grow inthe presence of pentachlorophenol. Thus, the introduction of the fungiinto the environment provides no new, non-naturally occurring organisms.

[0017] Another advantage is that the preparation and use of the fungalinoculum is fairly simple and utilizes agricultural waste products andwaste products from saw mills and urban chipping. These products havethe additional advantage of providing a quick and low cost food sourcefor the fungus, while having the added effect of stimulating rapid andextensive fungal growth, as well as providing a readily storable andtransportable solid matrix.

[0018] Another advantage is that the inoculum and its method of use areparticularly well suited for waste woods such as pressure-treated lumberfrom buildings, decks, utility poles and railroad ties. Specifically,the solid matrix of the fungal inoculum provides a wood environment forfungal growth which is similar to that of the waste wood. The fungalstrain is, therefore, readily adapted to the waste wood environment uponinoculation and does not require a period of adjustment beforedegradation and bioremediation begins.

[0019] These and other objects and advantages of the invention arereadily understood in view of the following detail description andexamples.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Waste wood containing pentachlorophenol (penta) is degraded andremediated in accordance with the present invention by inoculating thewaste wood with a fungal inoculum comprising at least one penta-tolerantfungus, a lignocellulosic substrate and a nutrient supplement. Thefungal inoculum is applied to the waste wood and maintained in anaerated and hydrated environment having temperature conditions and amoisture content sufficient to allow the inoculum to grow and at leastpartially remediate the penta or degrade the preservative treated wood.The inoculum and the waste wood are combined until a degradation productis achieved that is either of a volume consistent with the desires ofthe practitioner or capable of being recycled and used for paper orother composite woods.

[0021] Penta-tolerant fungi according to the present invention aregenerally defined as fungi capable of surviving and sustaining growthwhile being exposed to pentachlorophenol. In the preferred embodiment,the penta-tolerant fungi include, without limitation, Meruliporiaincrassata (Mad-563), Antrodia radiculosa (FP-103272-sp), Antrodiaradiculosa (L-11659-sp) and Antrodia radiculosa (FP-90848-T). Mostpreferably, the fungus utilized is Antrodia radiculosa (L-11659-sp). Thepenta-tolerant fungi, however, may also include any other penta-tolerantfungus capable of degrading or bioremediating the treated waste wood.Preferably, the chosen fungus should provide a two and one-half percent(2.5%) or more dry weight loss in the waste wood after about ten weeksof reaction time.

[0022] In the preferred embodiment, the penta-tolerant fungi arenaturally existing fungi not genetically altered or conditioned to growunder specific conditions or in the presence of a particularpreservative. It is anticipated, however, that one skilled in the artmay use a genetically altered or conditioned fungus in accordance withthe present invention. Genetically altered or conditioned fungi mayinclude, but are not limited to, any fungus modified to grow in thepresence of a specific nutrient supplement or food source.

[0023] In accordance with the Budapest treaty, the strains Meruliporiaincrassata (Mad-563), Antrodia radiculosa (FP-103272-sp), Antrodiaradiculosa (L-11659-sp) and Antrodia radiculosa (FP-90848-T) weredeposited with the Agricultural Research Culture Center (NRRL), anInternational Depositary Authority located at 1815 North UniversityStreet, Peoria, Ill. 60604 U.S.A., on Jul. 30, 1999, and given theaccession numbers NRRL 30171, NRRL 30168, NRRL 30167, and NRRL 30166,respectively.

[0024] The lignocellulosic substrate serves as a long-term food sourcefor the fungal inoculum as well as a matrix for its storage andhandling. Generally, “lignocellulosic substrate” refers to a substratehaving lignin, cellulose, or a combination of both lignin and cellulose.In the preferred embodiment the lignocellulosic substrate includessawdust or wood chips, either alone or in combination, but may alsoinclude any substrate that is capable of sustaining the growth of thefungi. Other lignocellulosic substrates may include, without limitation,agricultural residues such as rice straw, corn stalk, wheat straw, etc.

[0025] Sawdust or wood chips are preferred, however, for severalreasons: (1) they provide a long-term food source for the fungus whileproviding fungal growth in a wood environment similar to thepenta-treated waste wood environment experienced during inoculation; (2)they permit the production of a large quantity of fungi in a singlecontainer; (3) they provide a substrate for easily storing andtransporting the fungi; (4) they provide a matrix for convenient andeven distribution of the fungus at the inoculation site; and (5) theyprovide a low cost use of a waste product from saw mills.

[0026] The nutrient supplement is defined as a supplement for thelignocellulosic substrate which provides a food source that stimulatesrapid and extensive fungal growth beyond that obtained from thelignocellulosic substrate alone. The nutrient supplement may be of anyfood source which accomplishes the above stated goal and may differdepending upon the fungus selected. Preferably, however, the nutrientsupplement used is either a corn steep liquor, cornmeal or wheatbran.

[0027] Preferably, the fungal inoculum is prepared by first growing thepenta-tolerant fungus, or fungi, in culture containing malt extractagar. The culture is then typically incubated for one to two weeks, oruntil a confluent fungal growth is achieved over the agar surface. Theincubation is best performed in dark, aerobic conditions, at a relativehumidity of about 70%, and at a temperature range from about 20° C. to35° C., and more preferably at a temperature range from about 27° C. to32° C.

[0028] After achieving a confluent fungal growth, the fungus,lignocellulosic substrate and the nutrient supplement are combined toform the fungal inoculum, or “seeding.” The lignocellulosic substrate isfirst heat-sterilized and allowed to cool. The sterilizedlignocellulosic substrate is then mixed with the nutrient supplement andsterile water until a homogenous matrix is formed. The lignocellulosicsubstrate is preferably combined with the sterile water at 2-3 volumesof water per volume of substrate, while the nutrient supplement is addedin a range from about 1% to 5% per volume water. Liquid nutrientsupplement should always be added to the sterile water first and solidnutrient supplement should always be mixed with the substrate beforeadding the sterile water. This will ensure a homogenous matrix.

[0029] The homogenous matrix is gently mixed with the fungal culture andallowed to grow to form the final fungal inoculum. The mixture isallowed to grow for a time and under conditions which allow the fungusto reach confluent growth. Preferably, the mixture is grown in darkaerobic conditions, at a relative humidity of about 70%, and in atemperature range from about 20° C. to 35° C., and more preferably at atemperature range from about 27° C. to 32° C. Typically, confluentgrowth should occur within 4-8 weeks, but depends specifically upon thefungal volume introduced into the matrix, the lignocellulosic substrate,and the nutrient supplement. For example, a heavy fungal inocula withsawdust will shorten the period of fungal growth.

[0030] The fungal inoculum is ready for use or storage as soon as it hasreached confluent growth. If immediate use is desired, the inoculum canbe readily transported to the bioremediation and/or degradation sitewhere it is applied to completely cover the waste wood. In thealternative, if storage is desired, the fungal inoculum can be stored at4° C. The storage at 4° C. slows the fungal development and preventsovergrowth.

[0031] In one preferred embodiment of the present invention, largequantities of industrial inoculum can be produced either with largenumbers of tray inoculum, or in large durable plastic bags with aerationpatches to allow the appropriate airflow. Trays and bags can betransported easily to the field sites and applied on the waste wood.Production of inoculum directly in the truck or truckload container isalso a possibility.

[0032] In another embodiment, steam is an alternative source forsterilization. This is especially useful in pilot plants where steam isreadily available. In accordance with this embodiment, the steamprovides both sterility and moisture content for the lignocellulosicsubstrate. After being steamed, however, the substrate temperature mustbe cooled so as to avoid killing the fungi.

[0033] Once transported to the bioremediation/degradation site, thefungal inoculum is spread over the waste wood until all of the wastewood is covered. Preferably the waste wood is heat sterilized prior toapplication so as to minimize other environmental factors which mayeffect the ability of the fungi to properly degrade or bioremediate thewaste wood. Such factors typically include highly competitive bacteriaor other fungi.

[0034] The penta-containing waste wood and fungal inoculum mixture isthen aerated and hydrated for a time and under conditions sufficient toallow the inoculum to at least partially metabolize thepentachlorophenol and degrade the waste wood to a desired degradationproduct. The inoculated waste wood is maintained in a dark, aerobicenvironment, at a relative humidity of about 70%, and in a temperaturerange from about 20° C. to 35° C., and more preferably in a temperaturerange from about 27° C. to 32° C. The inoculation environment must haveample air space to ensure proper growth and to allow proper oxygen flow.In the absence of proper oxygen flow fungal growth will be hampered.

[0035] The degradation of the wood by the fungal inoculum will generallyresult in a degradation product having either a reduced volume or areduced concentration of pentachlorophenol Such a product will provide aresource capable of reuse in paper or wood composites, or simply have areduced volume such that storage or further processing is minimized.

[0036] The present invention is further explained by the followingexamples which should not be construed by ways of limiting the scope orspirit of the present invention.

EXAMPLES Example 1 Selection of Preservative-Tolerant Fungi

[0037] Fungal strains were collected after an extensive screening of thefungal library at the Center for Forest Mycology Research within theU.S. Department of Agriculture, Forest Products Laboratory. Thescreening process was first initiated by searching the library forfungal strains collected from specific wood products typically treatedwith wood chemical preservatives, i.e., utility poles, boats, decks,docks and railroad ties.

[0038] Other strains were isolated from wood samples collected from thefield plots of the USDA Forest Services, Forest Products Laboratory inGulfport, Mississippi and from Picnic Point in Madison, Wis. Theselected fungal strains were then retrieved and further analyzed todetermine their tolerance to CCA, ACQ, creosote and pentachlorophenol.

[0039] Chemical preservative tolerance was determined by application ofa “choice test.” The choice in this case was whether a particular fungalstrain would grow towards a wood treated with a chemical preservative ortowards a non-treated wood, or both.

[0040] To perform the “choice test”, a freshly grown fungal malt agardisk (9 mm) was placed in the center of a Petri dish (14 cm diameter)containing 12 ml of water agar, as described in Leithoff et al., “Growthof the copper tolerant ground-rot fungus Antrodia vaillantii ondifferent substrates,” The Int'l Research Group on Wood Preservation,IRG/WP95-10121, incorporated herein by reference. In this particularapplication, however, no glass ring was applied to the agar disk. Apreservative-treated wood sample (1.5 cm×3 cm) was then placed at oneedge of the Petri dish while a non-treated wood sample was placed at theopposite edge. The plates were then incubated at 27° C. and 70% relativehumidity (RH) for 14 days, wherein fungal growth was monitored.

[0041] At the end of 14 days, most fungal strains showed a primarygrowth preference towards the non-treated wood with no growth towardsthe treated wood. Some strains, however, showed growth preferencetowards both directions. Table 1 lists the strains which exhibited thegreatest amount of growth towards wood treated with pentachlorophenol.These fungal strains were considered penta-tolerant and selected for usein later degradation and bioremediation studies. TABLE 1 Fungi Selectedby Choice Test for Tolerance to Preservatives Isolate PreservativeStrain Name Designation Source Penta Phanerochaete chrysosporiumBKMF1767 FPL-MC Merulipoia incrassata Mad-563 FPL Irpex lacteus HHB7328FPL Polyporus sp FP101605 FPL-MC Phanerochaete sordida Kropp36T2 FPL-MCAntrodia radiculosa L-11659-sp FPL-MC Antrodia radiculosa FP-103272-spFPL-MC Antrodia radiculosa FP-90848-T FPL-MC

Example 2 Determination of Optimum Growth Conditions

[0042] The fungal strain Meruliporia incrassata (TFFH-294) was used todetermine the optimum growth conditions for fungal strains to be used infungal inocula effective in bioremediating and/or degrading chemicallypreserved wood. Fungal strain Meruliporia incrassata (TFFH-294) is astrain that was isolated from the USDA-FS Forest Products Laboratoryresearch plots in Gulfport, Miss. Meruliparia incrassata (TFFH-294) hasbeen deposited with NRRL and assigned accession number NRRL 30165.

[0043] Optimum Temperature

[0044] Four temperature settings, 20, 27, 32 and 37° C. were studied todetermine their effect on fungal growth. Four disks of fungal culturewere inoculated onto malt extract liquid medium (Difco Bacto maltextract). After incubation for 12 days at the various temperatures, themycelia of the fungi were harvested on Whatman No. 1 filter paper, airdried and measured for biomass dry weight.

[0045] The results showed that the optimal temperature for fungal growthwas in a range between about 27° C. and 32° C. Incubation at a highertemperature setting of 35° C. showed a substantial decline in growth, asdid incubation at a lower temperature setting of 20° C.

[0046] Optimal Light Conditions

[0047] Light effect on growth was studied under three settings, 24 hoursof light, 12 hours of light with 12 hours of darkness, and 24 hours ofdarkness. Fungal inoculant was grown in malt extract liquid medium asabove for a period of 21 days. Mycelia were then harvested, air driedand weighed to obtain biomass measurements.

[0048] The results of light effect are shown in Table 2. Fungi producedmore cell mass under the complete dark growth condition than undereither a mixture of light/dark or complete light. Cell mass measurementsincreased 33% at 24 hours dark cycle then at 24 hours light cycle. TABLE2 Light Effect on TFFH-294 Growth Light condition Dry Weights, mg(±S.D.) 24 hours of light 66 ± 8 12 hours of light/dark 70 ± 6 24 hoursof dark  88 ± 13

[0049] Defined Liquid Medium

[0050] Fungal cultures were inoculated in Bailey media (Bailey et al.,“Cellulase (B-1,4-Gucan,4-Glucanohydrolase) from Wood-Degrading FungusPolyporus Chweinitzii,” Fr. I. Purification Biochem. Biophys. Acta,185:381-391 (1969)), and BIII media (Kirk et al., “Production ofMultiple Ligninases by Phanerochaete chrysosporium: Effect of SelectedGrowth Conditions and Use of a Mutant Strain,” Enzyme Microb. Technol.,8:27-32 (1986)) to determine which medium provided better fungal growth.Fresh fungal cultures were grown on malt extract agar plates. Four agardiscs (9 mm) of complete fungal growth were removed and inoculated into25 ml growth medium in 125 ml flasks. The flasks were then keptstationary at 27° C. and 70% RH for 21 days. After 21 days, the gyceliawere harvested for biomass measurements.

[0051] The biomass measurements indicated that the biomass growth forTFFH 294 was better in Bailey medium over BIII medium. The Bailey mediumproduced an average dry weight mass of 20 mg±10 mg, while the BIIImedium produced an average dry weight mass of 16 mg±5 mg. It isunderstood, however, that certain types of fungi may grow better incertain types of media. Accordingly, the limitation of the presentinvention to one type of media over another is not necessary.

[0052] Oxygen

[0053] Fungal cultures were grown with or without oxygen-enhancement,i.e., oxygen flush, to determine the effects of oxygen upon fungalgrowth. Fungal cultures were inoculated in Bailey media or BIII media,and grown on malt extract agar plates. From the plates, four agar disks(9 mm) of complete fungal growth were removed and inoculated into 25-mlgrowth medium in 125-ml flasks. The flasks were grown with and withoutoxygen flush, and were kept stationary at 27° C. and 70% RH for 21 days.Oxygen-enhanced cultures were flushed with oxygen every other day for aninterval of 20 seconds. After 21 days, the fungal mycelia wereharvested, air dried and measure for dry weight.

[0054] From the mycelia dry weight measurements, the results showed thatoxygen-enhanced incubation provided fungal growth similar to that of thewhite-rot fungi, Phanaerochytes chrysoporium (Kirk. et al. 1986). Asseen in Table 3 below, the enhanced growth effect has consequentlyproduced a lower pH value in the culture flasks, thus indicatingconfluent growth. TABLE 3 Oxygen Effect on Fungal Growth with TwoDefined Media Strain Medium O₂ Dry weights, mg (± S.D.) pH TFFH 294Bailey + 23 ± 1 2.73 - 20 ± 1 3.04 BIII + 21 ± 2 2.96 - 18 ± 2 3.61

Example 3 Nutrient Supplements: Corn Steep Liquor

[0055] Fungal inoculum containing Meruliporia incrassata (TFFH-294) wasadded to malt extract liquid media supplemented with 1, 2.5 and 5% ofsterile corn steep liquor (CSL)(ADM corn processing, Cedar Rapids,Iowa). Media containing no CSL was used as a control. Cultures weregrown at 27° C. and 70% RH for 30 days. After 30 days, Mycelium wasseparated from liquid culture by filtration, oven dried and weighed. Thedry weight was determined as a percent of the dry weight for theinoculum without CSL concentration.

[0056] The addition of the corn steep liquor to the growth medium had aprofound effect on fungal growth. Various concentrations of CSL weretested and results are shown in Table 4 below. The highest enhancementeffect was obtained by adding 1% CSL in malt extract medium. Thisenhancement should limit the disadvantage experienced by various fungalstrains such as TFFH-294, which are typically disadvantaged by theirslow growth characteristic that limits their competition with otherdominant fungi in nature, such as Postia placenta and Gloeophyllumtrabeum. Accordingly, the supplementation with a CSL nutrient will allowbetter competition in nature. TABLE 4 Effect of CSL on TFFH-294 GrowthCSL concentration % Dry weight gain 0      100 1.0% 321 2.5% 256 5.0%196

Example 4 Effect of Corn Steep Liquor on Fungal Growth for FungiTolerant to Different Wood Chemical Preservatives

[0057] Fungal inoculum containing fungal strains exhibiting tolerance toeither CCA, ACQ, creosote or pentachlorophenol were supplemented with 1%sterile CSL (ADM corn processing, Cedar Rapids, Iowa) in malt extractmedia to determine the effect of the CSL on the growth patterns of theseparate strains. Media without CSL was used as a control and consideredto exhibit 100% growth. Weight was determined from fungal myceliaincubated for 3 weeks in liquid malt extract, with or without CSL, at27° C. and 70% RH. After 3 weeks, the mycelia was removed from theliquid by filtration on filter paper, oven dried and weighed. The % dryweight was determined as percent of the dry weight for the inoculumwithout CSL. Table 5 illustrates the increase growth effect that the CSLprovides to the various strains used. TABLE 5 Effect of corn steepliquor (CSL) on fungal growth in liquid medium. Strain* CSL Net weight(g){circumflex over ( )} % growth{circumflex over ( )}{circumflex over( )} TFFH-294 -- 0.053 ± 0.011 100 + 0.108 ± 0.027 203 FP-90848-T --0.079 ± 0.013 100 + 0.116 ± 0.023 147 Mad-534 -- 0.049 ± 0.004 100 +0.114 ± 0.006 233 Mad-617 -- 0.028 ± 0.003 100 + 0.106 ± 0.022 379FP-103272-sp -- 0.058 ± 0.009 100 + 0.114 ± 0.017 196

Example 5 Preservative Wood Degradation Study

[0058] A degradation study was performed using fungal strains exhibitingtolerance to either CCA, ACQ, creosote or pentachlorophenol. Blocks ofwood were cut (lxlxO.3 inches) from southern pine (Pinus sp.) andtreated to CCA, ACQ, creosote and pentachlorophenol according toAmerican Wood Preserver's Association (AWPA) standards (“American WoodPreserver's Association: Book of Standards,” American Wood-Preserver'sAssociation, Woodstock, Md., 1991). The blocks of wood were sterilizedand allowed to cool.

[0059] Degradation of the treated wood block was observed by placing thesterile block on the surface of the fungal inoculum in the glass bottle.Fungi strains known to be tolerant to a specific preservative wereexposed to blocks having their respective preservative. The blocks werethen incubated for 10 weeks at 27° C. and 70% RH. After 10 weeks, theremaining portion of the blocks were removed and their dry weight losswas measured according to ASTM standards.

[0060] Table 6 below depicts the ability of the fungal inoculum havingcertain fungal strains to degrade the preservative-treated wood. As canbe seen the fungal strains of Meruliporia incrassata (TFFH-294),Antrodia radiculosa (MJL-630), Meruliporia incrassata (Mad-563) andAntrodia radiculosa (FP-90848-T) were able to degrade the Penta-treatedwood at an average of greater than 20% of the original dry weight of thewood. Antrodia radiculosa (FP-90848-T) was also able to degradeACQ-treated wood at an average of 29.9% of the original dry weight.Antrodia radiculosa strains FP-103272-sp, L-11659-sp, and FP-90848-T,and Meruliporia incrassata (Mad-563) were able to degrade on averageapproximately 3% of the wood block having a concentration ofPentachlorophenol. Finally, Antrodia radiculosa strains FP-103272-sp,L-11659-sp, and FP-10539-R, and Neolentinus lepideus (Mad-534) exhibitedthe ability to reduce the dry weight of wood having creosote by anaverage of approximately 3%. TABLE 6 Fungal Degradation ofPreservative-Treated Wood* UNTREATED ACQ CCA Penta Creosote Fungus AvgSD Avg SD Avg SD Avg SD Avg SD Meruliporia incrassata (TFFH-294) 62.22.9 9.7 5.7 36.8 2.7 1.9 0.3 1.8 0.2 Antrodia radiculosa (MJL-630) 32.64.8 6.7 6.8 26.6 2.9 1.5 0.1 1.7 0.2 Meruliporia incrassata (Mad-563)62.5 2.5 3.5 0.1 23.7 7 4.1 2.5 1.5 0 Antrodia radiculosa (FP-90848-T)39.5 4.1 29.9 14.3 20.1 7.7 2.6 0.5 2.1 0.2 Antrodia radiculosa(FP-103272-sp) 24.6 6 0.7 0.1 6.5 4.7 4.7 2.3 5.5 2 Antrodia radiculosa(FP-105309-R) 27.2 3 4.4 4 2.3 0.8 2.4 0.6 2.9 0.8 Antrodia radiculosa(L-11659-sp) 23.1 2.7 0.8 0.3 1.3 1.3 5.3 1.8 3.2 1.8 Neolentinuslepideus (Mad-534) 38.8 5.3 1.4 0.3 −0.7 0.4 1.5 0.1 4.1 0.7

[0061] Effect of Additives on Degradation of Preservative-Treated Wood

[0062] A study was also performed to determine what effect the additiveshad on the ability of certain fungal strains to degradepreservative-treated wood. A fungal inoculum was prepared for eachstrain using one of the selected fungi as described above. First, thefungus was grown on 10 ml malt extract agar in a glass bottle (2×2×5 hinches) and incubated at 27° C., 70% RH, for 1-2 weeks until a confluentgrowth on the agar layer occurred. A mixture of soft wood and hard woodsawdust was sterilized and set for use as a lignocellulosic substrate.

[0063] Separate fungal inocula were prepared containing a nutrientsupplement of either corn steep liquor or cornmeal and wheatbran. Forthe inoculum containing corn steep liquor, 10 g of the sterile sawdustwas mixed with 20 ml of sterile water containing 1% commercial cornsteep liquor (v/v) and added to the glass bottle containing the fungalgrowth. For the inoculum containing cornmeal and wheatbran, 10 g of thesterile sawdust was combined with the corn meal and wheat bran at 2.5%(w/w, 0.25 g corn meal or wheat bran/10 g sawdust) for each ingredient,followed by 20 ml of sterile water. The inoculum was then added toanother glass bottle containing the fungal growth. Incubation wascontinued in a stationary condition and at a temperature of 27° C. and70% RH. The length of incubation depended on the rate of fungal growth,however, after about 4-6 weeks of incubation fungal mycelia growth wasobvious. The fungal strains utilized are those depicted in table 7.These results indicate that additives enhance degradation ofpreservative-treated wood. TABLE 7 Effect of additives to degradation ofpreservative-treated wood. CHEMICAL FUNGAL SPECIES ISOLATE# WEIGHT*cmwb** CSL*** SYP**** CCA Antrodia radiculosa L-11659 sp 32.0 mg 150%446% 972% Polyporus sp FP134933 12.3 mg 1040%  577% 1284%  (unknown)F43G  1.0 mg 3500%  500% 114% Diplomitoporus lindbladii FP134600 13.0 mg469% 238% 753% Meruliporia incrassata TFFH 294 34.0 mg  32% 208% 179%ACQ Chain chlamydospore ME681 27.0 mg  63%  56% 248% Antrodia radiculosaFP90848 17.0 mg 176% 188% 243% — UpK 38.0 mg 118% 103%  34% — UpL 50.0mg  82% 120% 678% Creosote Gloeophyllum FPL 508 64.0 mg 103%  39%Subferrugineum Melanoporia niger MD278 66.0 mg  95% 127% 772% — UpK 51.0mg 112% 110%  14% Polyporus sp. FP101605 61.0 mg 101% 111% 224%

Example 6 Scale-up of Fungal Inoculum in Laboratory

[0064] Fungal inoculum containing Meruliporia incrassata (TFFH-294) wasprepared according to the present invention and utilized to determinethe effectiveness of the fungal inoculum on a larger scale. Although thefungal incoculum employed was directed towards wood treated with CCA, itis anticipated that similar results would be obtained on wood treatedwith pentachlorophenol using penta-tolerant fungal strains.

[0065] Fungal Culture Preparation

[0066] Five to seven Petri dishes (14 cm diameter) containing malt agarwere used to grow fungal inoculum containing the fungal strainMeruliporia incrassata (TFFH-294). The Petri dishes were incubated at27° C. and 70% RH until a confluent growth occurred on the agar layer.Agar chunks of 1-1.5 inches square were removed and immediatelytransferred to the solid substrate matrix already prepared as describedbelow.

[0067] Solid Substrate Matrix in Tray

[0068] Solid substrate comprising a lignocellulosic substrate andnutrient supplement were prepared in an aluminum tray (9×13×2.5 hinches). First, 350 gm of sawdust was placed in the empty tray and thetray and sawdust autoclaved and cooled to room temperature. Once thetray and sawdust was cooled, 700 ml of sterile water having a 1%concentration of corn steep liquor was added and mixed to achieve ahomogenous solid matrix. The fungal squares were then gently mixed withthe solid matrix, covered with foil and incubated. Incubation was indark conditions and at 27° C., 70% RH, for a period of 4-8 weeks, oruntil a confluent growth was obtained. After the fungus had reachedconfluent growth, the fungal inoculum was stored at 4° C. to preventovergrowth.

[0069] Preservative Lumber Degradation in Degradation Chamber

[0070] Several large metal degradation chambers (33×6×8 h inches) withsliding covers were custom-made for the lumber degradation study. A 2inch layer of moistened soil having a water content of 35% was placed inthe degradation chamber. Formed pieces of 12 inch cutpreservative-treated 2×4 lumber was pieced on top of the soil and themetal degradation chambers were sterilized in an autoclave. After thechambers had cooled down to room temperature, the TFFH-294 fungal chipsinoculum was poured onto the preservative-treated lumber until thelumber was completely covered. The chamber containing the inoculatedlumber was then stored at 27° C. and 70% RH for 12 weeks. At the end ofthe 12 week incubation period the wood was harvested and measured fordry weight loss to determine the level of degradation. Results showed a28% degradation of CCA-treated wood as compared to nontreated controlwood.

[0071] While the present invention has now been described andexemplified with some specificity, those skilled in the art willappreciate the various modifications, including variations, additions,and omissions, that may be made in what has been described. Accordingly,it is intended that these modifications also be encompassed by thepresent invention and that the scope of the present invention be limitedsolely by the broadest interpretation that lawfully can be accorded theappended claims.

We claim:
 1. A method for bioremediating wood containing chromatedpentachlorophenol (penta) comprising the steps of: inoculating woodcontaining pentachlorophenol with a fungal inoculum comprising at leastone penta-tolerant fungus, at least one lignocellulosic substrate and atleast one nutrient supplement, the lignocellulosic substrate and thenutrient supplement in an amount sufficient to produce a biomass of thefungal inoculum sufficient to at least partially remediate thepentachlorophenol; and aerating and hydrating the inoculated wood for atime and under conditions sufficient to allow the fungal inoculum toremediate the pentachlorophenol.
 2. The method of claim 1 wherein thepenta-tolerant fungus is selected from the group consisting ofMeruliporia incrassata (Mad-563), Antrodia radiculosa (FP-103272-sp),Antrodia radiculosa (L-11659-sp) and Antrodia radiculosa (FP-90848-T).3. The method of claim 1 wherein the penta-tolerant fungus is Antrodiaradiculosa (L-11659-sp).
 4. The method of claim 1 wherein thelignocellulosic substrate is selected from the group consisting ofsawdust, wood chips, rice straw, corn stalks, and wheat straw.
 5. Themethod of claim 1 wherein the nutrient supplement is selected from thegroup consisting of corn steep liquor, cornmeal and wheatbran.
 6. Themethod of claim 1 wherein the inoculated wood is aerated and hydrated indark, aerobic conditions, at a relative humidity of about 70%, and in atemperature range from about 20° C. to 35° C.
 7. The method of claim 6wherein the inoculated wood is aerated and hydrated in a temperaturerange from about 27° C. to 32° C.
 8. A method for degrading woodcontaining pentachlorophenol (penta) comprising the steps of:inoculating wood containing pentachlorophenol with a fungal inoculumcomprising at least one penta-tolerant fungus, at least onelignocellulosic substrate and at least one nutrient supplement, thelignocellulosic substrate and nutrient supplement in an amountsufficient to produce a biomass of the fungal inoculum sufficient todegrade the wood; and aerating and hydrating the inoculated wood for atime and under conditions sufficient to allow the fungal inoculum todegrade the wood to reach a degradation product.
 9. The method of claim8 wherein the penta-tolerant fungus is selected from the groupconsisting of Meruliporia incrassata (Mad-563), Antrodia radiculosa(FP-103272-sp), Antrodia radiculosa (L-11659-sp) and Antrodia radiculosa(FP-90848-T).
 10. The method of claim 8 wherein the penta-tolerantfungus is Antrodia radiculosa (L-11659-sp).
 11. The method of claim 8wherein the lignocellulosic substrate is selected from the groupconsisting of sawdust, wood chips, rice straw, corn stalks, and wheatstraw.
 12. The method of claim 8 wherein the nutrient supplement isselected from the group consisting of corn steep liquor, cornmeal andwheatbran.
 13. The method of claim 8 wherein the inoculated wood isaerated and hydrated in dark, aerobic conditions, at a relative humidityof about 70%, and in a temperature range from about 20° C. to 35° C. 14.The method of claim 13 wherein the inoculated wood is aerated andhydrated in a temperature rage from about 27° C. to 32° C.
 15. Themethod of claim 8, wherein the degradation product exhibits a loss indry weight of an amount in excess of 2.5% of the dry weight of the wood.16. A fungal inoculum for degrading and bioremediating wood containingpentachlorophenol (penta) comprising at least one penta-tolerant fungus,at least one lignocellulosic substrate, and at least one nutrientsupplement.
 17. The fungal inoculum of claim 16 wherein thepenta-tolerant fungus is selected from the group consisting ofMeruliporia incrassata (Mad-563), Antrodia radiculosa (FP-103272-sp),Antrodia radiculosa (L-11659-sp) and Antrodia radiculosa (FP-90848-T).18. The fungal inoculum of claim 16 wherein the penta-tolerant fungus isAntrodia radiculosa (L-11659-sp).
 19. The fungal inoculum of claim 16wherein the lignocellulosic substrate is selected from the groupconsisting of sawdust, wood chips, rice straw, corn stalks, and wheatstraw.
 20. The fungal inoculum of claim 16 wherein the nutrientsupplement is selected from the group consisting of corn steep liquor,cornmeal and wheatbran.
 21. A method for preparing a fungal inoculum fordegrading or bioremediating wood containing pentachlorophenol (penta),comprising the steps of: combining at least one penta-tolerant funguswith a matrix comprising at least one lignocellulosic substrate, atleast one nutrient supplement, and sterile water; and growing thepenta-tolerant fungus and matrix combination in dark, aerobicconditions, at a relative humidity of about 70%, and in a temperaturerange of about 20° C. to 35° C.
 22. The method of claim 21 wherein thepenta-tolerant fungus and matrix combination is grown in a temperaturerange of about 25° C. to 32° C.
 23. The method of claim 21 wherein thevolume of lignocellulosic substrate is about 25% to 33% the volume ofsterile water, and the volume of nutrient supplement is about 1% to 5%the volume of sterile water.